Melanoma and prostate cancer specific antibodies for immunodetection and immunotherapy

ABSTRACT

Methods and compositions are provided for detecting antigens having a specific epitope associated with melanoma and prostatic carcinoma. The epitope is present in melanoma cells and prostatic cancer cells but is essentially absent from melanocytes and normal prostatic tissue. The antibody can be used in diagnostic methods for histochemical detection of human melanoma and prostate carcinoma, of various progression stages and in treatment of melanoma and prostate carcinoma.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. Ser. No.08/654,641, filed May 28, 1996, now allowed, which is acontinuation-in-part U.S. Ser. No. 07/829,855 filed Jan. 31, 1992, nowU.S. Pat. No. 5,605,831, which disclosures are herein incorporated byreference

FIELD OF INVENTION

[0002] The subject invention is related to the use of antibodies, whichbind to a unique peptide obtainable from a Xiphophorus melanomamrk-receptor tyrosine kinase for the diagnosis and therapy of melanomaand prostate cancer.

BACKGROUND

[0003] The ability to detect and diagnose cancer through theidentification of tumor markers is an area of widespread interest. Tumormarkers are substances, typically proteins, glycoproteins,polysaccharides, and the like which are produced by tumor cells and arecharacteristic thereof. Often, a tumor marker is produced by normalcells as well as by tumor cells. In the tumor cells, however, theproduction is in some way atypical. For example, production of a tumormarker may be greatly increased in the cancer cell. Additionally, thetumor marker may be released or shed into the circulation. Detection ofsuch secreted substances in serum may be diagnostic of the malignancy.Therefore, it is desirable to identify previously unrecognized tumormarkers, particularly, tumor markers which are secreted into thecirculation and which may be identified by serum assays It is alsodesirable to develop methods and compositions which allow determinationof the cellular origin of a particular tumor or other proliferativedisease, for example by radioimaging techniques The location of thetumor markers on the surface of the cells, particularly where there isan extracellular domain that is accessible to antibodies (i.e., thedomain acts as a receptor for the antibodies), provides a basis fortargeting cytotoxic compositions to the receptor. Examples ofcompositions of interest in such a method include complement fixingantibodies or immunotoxins which bind to the receptor as a means ofspecifically killing those cells which express the receptor on the cellsurface.

[0004] Human malignant melanoma arises from a series of stages startingwith the harmless mole, going through intermediate stages of radial toinvasive growth and ending in the destructive final stage of metastaticmelanoma. Melanoma usually resists chemotherapy as well as radiotherapy.Surgery is the most effective treatment. However, for it to beeffective, surgery requires early diagnosis which is unfortunatelyhampered by the lack of accurate markers for melanoma. Melanomaassociated antigens have been found, but they are of little diagnosticvalue. For example, the nerve growth factor receptor is found in highdensity on melanoma cells. However, monoclonal anti-nerve growthreceptor antibodies are specific for neural crest cell diseases ratherthan for melanoma alone. Likewise, other melanoma associated antigensagainst which antibodies have been raised are nonspecific for melanomacells. Examples are the monoclonal antibodies raised against in vitrogrown melanoma cells which are directed against gangliosides orglycoproteins present on the melanoma cells. Both antigens are alsofound on other cells.

[0005] Adenocarcinoma of the prostate is one of the most common tumorsin men and accounts for 10% of deaths from malignant disease in males inthe United States. Only a small proportion of these cases becomesclinically apparent prior to death, the remainder being latentcarcinoma. Radical prostatectomy remains the treatment of choice fortumors confined to the gland but this is applicable to only a tinyfraction of cases. Orchiectomy and hormone therapy (usually estrogentherapy) together appear to be the most effective palliative treatmentin patients with symptomatic cancer of the prostate and are also used asan adjunct to surgery. However, there are significant side effects tothe use of estrogens, including an increase in mortality fromcardiovascular disease.

[0006] Three-fourths of the tumors arise in the posterior lobe, andurinary symptoms therefore tend to occur late in the disease Theidentification and isolation of cancer genes, most notably demonstratedfor colon carcinoma, has been a major breakthrough for our understandingof tumorigenesis. Cancer is basically a disease of multiple geneticchanges resulting in stages of progression of a normal cell into ahighly malignant, metastasizing cell.

[0007] However, very little is known about the development and clinicalprogression of prostatic carcinoma at the genetic level. Studies offamilial clustering of prostate cancer have provided evidence for arare, high risk autosomal dominant allele which may be responsible forearly onset of prostate cancer (Carter B. S., et al., Proc. Natl. Acad.Sci USA 89:3367-3371, 1992; Carter B. S., et al., J. Urol. 150:797-802,1993). Indeed a consortium of several research groups has recentlylocalized a major prostate cancer susceptibility locus on the long armof chromosome 1 (1q24-25) (Smith R. J et al., Science 274:1371-1373,1996). In addition, several chromosomal alterations such as gain of 8qand loss of 8p (Visakorpi T. et al., Cancer Res. 55:342-347, 1995; cherM. L. et al., Cancer Res. 56:3091-3102, 1996) as well as loss of 10q,13q, 16q, 17p (Isaacs W. B., et al., Cancer Surveys 23.19-32, 1995; CherM. L. et al., Cancer Res. 56:3091-3102, 1996) and 11p11.2 (Dong, J. T.,et al., Science 268:884-886, 1995) have been identified. Similarly, thegroup at the Mayo Clinic (Qian et al., 1995) using fluorescence in situhybridization has shown gains of chromosome 7, 8, 10 and 12 and detectedsimilar proportions of anomalies in PIN and carcinoma supporting thenotion that PIN is the supposed precursor lesion. This study showed alsothat the gain of chromosome 8 was the most common alteration and mostlycorrelated with the cancer grade. This amplification of chromosome 8genes may lead to overexpression and could play a key role inprogression of prostatic carcinoma.

[0008] A significant number of patients come into the physician withsymptoms due to distant metastases. Cancer of the prostate arisesthrough a continuum from normal luminal secretory cells which progressthrough a dysplastic stage of mild to severe prostatic intraepithelialneoplasia (PIN) to carcinoma in situ and invasive carcinoma cells. PINis considered the precursor stage; however, once grade 3 is reached, thecancer enters a malignant stage which is characterized by cells breakingloose from the epithelium and invading the neighboring stromal componentof the prostate gland. This invasion takes place where the basal celllayer is disrupted and the basement membrane is fragmented. The cancercells then progress from well. to moderately and finally poorlydifferentiated cells. These cells, staged according to Gleason grades1-5, are believed to reflect the increasing aggressiveness of the cancerof the prostate.

[0009] Frequent routine rectal examinations are the best means ofdemonstrating early and operable prostatic tumors. Measurement ofprostate specific antigen as a screening test for prostate cancer hasbeen used but presents both technical difficulties and a high falsepositive rate. Prostatic acid phosphatase also has been used as a markerfor prostate cancer but does not detect all cancers. The most successfuldetection of prostate cancer is from the combined use of a digitalrectal exam, transrectal ultrasound and detection of prostate specificantigen. The sensitivities of the three tests individually vary from 50%to 85% but the positive predictive value fluctuates around 30%. Whenthese three investigations are summated, the detection rate isapproximately twice as high as when a single parameter is used. The onlyreliable procedure for definitive diagnosis of prostatic carcinoma is byopen perineal biopsy. Needle biopsies and cytologic studies of prostaticfluid are unreliable for the diagnosis of early cancer but are usefulmethods of obtaining a histological diagnosis in the more advancedcases. It therefore is of interest to identify in particular, earlystage prostatic cancer and to identify non-invasive methods of treatingprostatic cancer. It also is of interest to identify amelanoma-associated antigen which is specific for melanoma as comparedto normal melanocytes as well as other normal and malignant cells. Anantibody raised against such an antigen can be used in the diagnosis andtreatment of melanoma. The antibody itself or an immunotoxin may finduse as an antiproliferative agent. So far no single gene has been shownto be implicated in the genesis of prostate carcinoma. Once such genesare identified and characterized this will provide important insightinto the development of this disease. In a larger frame work, it maythen be possible to identify individuals at high risk and to predictwhether or not an indolent tumor has the potential to become malignant.This may lead to new avenues for treatment of the disease.

[0010] Relevant Literature

[0011] U.S. Pat. No. 4,590,071 is directed to a cytotoxic conjugatespecific for human melanoma. Maguire, et al., Cancer (1993) 72:(11Suppl.) 3453-62, disclose use of an antigen expressed by the majority ofadenocarcinomas for preparation of immunoscintigraphic agents for thepreclinical staging of prostatic carcinoma in patients with negative orequivocal results on standard imaging tests. Lopes, et al., CancerResearch (1990) 50:6423-9, disclose a prostate-reactive monoclonalantibody. Horoszewicz (U.S. Pat. No. 5,162,504), discloses monoclonalantibodies to an antigen on prostatic epithelial cells. U. S. Pat. No.5,605,831 discloses a monoclonal antibody which binds to an epitopewhich is present on human melanoma cells and absent from melanocytes andmalignant cells other than melanoma and prostatic cancer cells.

[0012] In the Xiphophorus fish melanoma model several genetic loci havebeen identified which mediate melanoma formation (see Vielkind, J. R.(1992) in Transformation of Human Epithelial Cells: Molecular andOncogenic Mechanisms, Milo G E, Casto B C, Shuler C F, (eds), CRCPress). A duplicated gene, Xmrk that genetically maps to an oncogeniclocus has been cloned. The gene encodes a novel growth factor receptortyrosine kinase which has similarities to the epidermal growth factorreceptor (EGFR); its expression correlates with active melanoma growth(Woolcock et al., (1994) Cell Growth Diff 5:575-583). Overexpression ofXmrk results in autophosphorylation of the Xmrk protein in fishmelanomas and cultured melanoma cells as well as in cultured fish, mouseand human cells transfected with a high expression CMV-Xmrk construct(Wittbrodt et al., (1992) EMBO J. 11:4239-4246).

SUMMARY OF THE INVENTION

[0013] The present invention provides methods and compositions usefulfor detecting or monitoring primary and metastatic melanomas andprostatic carcinomas. The methods use an antibody which is specific forepitope on the surface of melanoma and prostatic cancer cells. Theantibody is produced using an immunogen comprising a unique sequencederived from a Xiphophorus melanoma. The epitope can be detected on thecarcinoma cells in tissue biopsies, in carcinoma cells in culture, aswell as in blood samples. The epitope appears to be located on thesurface of the melanoma and prostatic carcinoma cells and may be a cellsurface receptor. The putative receptor has an apparent molecular weightof 170 KD by western blotting of lysates of a melanoma cell line.Detection of the epitope conveniently is accomplished by reaction withmonoclonal antibodies (XMEL) derived from hybridoma cell line 12f3.2, orantibodies having a similar specificity, and detecting the formation ofspecific antigen-antibody complexes. The antigens and/or antibodies finduse in vitro or in vivo in diagnosis, prognosis and therapy.Antiidiotypic antibodies and the antigenic peptide also find use fordetecting the presence of antibodies to the antigen in the blood orserum of a human host.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014]FIG. 1. FIG. 1A shows a schematic structure of putative mrkreceptor tyrosine kinase, SP=signal peptide, TM=transmembrane domain,TK=tyrosine kinase domain, striped boxes=cystine-rich domains, belowsimilarities of various domains to the human epidermal growth factorreceptor HER- 1 (Wittbrodt. et al., (1989) Nature 341:415-421); FIG. 1Bshows surface values of amino acid residues 340-640 in which the peptidespans amino acids 480-489. FIG. 1C shows the nucleotide sequence (SEQ IDNO: 2and amino acid sequence (SEQ ID NO: 3) spanning the amino acidsequence from 446-486.

[0015]FIG. 2 shows immunostaining of cross-sections from a commonacquired nevus FIGS. 2A and 2B); a primary (vertical growth phase)melanoma (FIGS. 2C and 2D); and melanomas metastatic to the skin withthe monoclonal antibody XMEL (FIGS. 2E and 2F and FIGS. 2G and 2H). FIG.2A: overview, 25x; FIG. 2B: 250x of inset in FIGS. 2A showing weak,diffuse red positive staining of nevocytic cells with occasionalpunctate staining; FIG. 2C: overview, 25x, strong red staining in upperand lower areas of primary melanoma, weaker staining in the more centralarea, FIG. 2D: 250x of inset in FIG. 2C showing the strong, granularstaining of nests of primary melanoma cells; FIG. 2E. overview, 25x,staining appears to be stronger on the periphery than in the centralpart of the metastatic tumor, FIG. 2F 250x of inset in FIG. 2E showingdiffuse staining of variable intensity on melanoma cells: FIG. 2G:overview, 25x, showing areas of the metastatic melanoma with variablebut weak staining, FIG. 2H: 250x of inset in FIG. 2G showing the weakdiffuse and variable staining of the melanoma cells, occasionally apunctate staining can be seen. In all the sections from the variousspecimens no staining was observed on other cells including normalmelanocytes. Immunostaining was performed with ABC peroxidase stainingkit (Vector Lab.) and AEC (3-amino-9-ethylcarbazole, Sigma) as thechromogenic substance; sections were counterstained with Mayer'shematoxylin.

[0016]FIG. 3 shows a western blot analysis of proteins from cells ofXiphophorus PSM (lane 1,3) and human KZ13 melanoma cell line (lane 2).Proteins in lanes 1 and 2 were immunostained with XMEL hybridomasupernatant, rabbit anti-rat IgG(H+L) secondary antibody (Sigma) and DAB(3,3′-diaminobenzidine, Sigma) as chromogenic substance; proteins inlane 3 were reacted with hybridoma growth medium instead of XMELsupernatant. The XMEL monoclonal antibody stained faintly protein bandsof 170,000, 115,000 and 60,000M_(r) (closed triangles) in lysates fromthe PSM cells (lane 1) and stained faintly protein bands of 150,000 and50,000M_(r) (open triangles) in lysates from the KZ13 human melanomacell line (lane 2) The strong protein band which appears in all 3 lanesmost likely represents cross-reactivity with the growth medium and/orsecondary antibody as it also appears in proteins from the PSM cellswhen the XMEL supernatant is replaced with the growth medium in theimmunostaining reaction lane 3). Numbers on the right side representM_(r)×10³ of rainbow protein marker (Bio Rad)

[0017]FIG. 4 shows XMEL immunostaining of prostatic carcinoma. FIG. 4Ashows that a high grade PIN reacts strongly with the XMEL MoAb, whilethe benign gland (arrow) is negative, as is the surrounding stromalcomponent. Microscopically, this area of PIN demonstrates typicaldysplastic features: enlarged nuclei, prominent nucleoli and increasednumber of cells. FIG. 4B shows prostatic cancer cells infiltratingaround a normal gland (arrow). FIG. 4C shows positive staining ofprostate cancer cells on membranes. FIG. 4D shows typical invasionaround a nerve (N) by a prostate cancer positive for XMEL MoAb binding.

DESCRIPTION OF SPECIFIC EMBODIMENTS

[0018] In accordance with the subject invention, methods andcompositions are provided for the detection, identification, monitoringand treatment of carcinomas, especially melanomas and prostatic tumors,including high grade prostatic intraepithelial neoplasia (PIN). Inmelanomas, an about 170 KD protein is present on the cell surface of thecancer cells which is immunologically related to an mrk-receptortyrosine kinase present in the Xiphophorus fish melanoma. Patientsamples are screened for tumor cells by assaying for the presence ofthis cell surface protein in patient samples, including tissue biopsyspecimens and blood samples; the receptor apparently is “shed” into thebloodstream.

[0019] Current clinical methods of predicting pathologic tumor stage ofprostatic carcinoma, preoperatively, are extremely limited. Criteriasuch as pathologic stage, PSA level, histologic grade (well and poorlydifferentiated cancer cells, Gleason grade), tumor volume and percent oftumor are important prognosticators; however, they do not absolutelypredict outcome of the tumor for the individual patient (see Humphrey,P. A., Walther P. J., Am. J. Clin. Pathol 100:256-269, 1993).Furthermore, determination of DNA content in prostatic cancer cells byflow cytometry also does not seem to predict clinical outcome (Jones E.C. J. Urol 144:89-93, 1990). Thus, although histopathologicalprognosticators are helpful, they only indicate trends and likelihood ofthe outcome. The current invention offers the advantage that it providesvaluable prognostic information not available from current clinicalmethods. Because the 170 KD protein is on the cell surface, it isreadily accessible to an antibody for detection of cancer cells in atissue sample or for treatment of cancer cells. Also, it is easier for asurface protein than a cytoplasmic protein to shed into the bloodstream, which provides a serum marker for detection of cancer. A serumassay for detecting of a cancer marker is a non-invasive method, whichis more acceptable to patients and also provides a tool for screeninglarge number of samples. Additional advantages include that the antibodyrecognizes an antigen that is related to the early events (primarymelanomas) rather than the later stages of progression to the metastaticphenotype (metastatic melanomas). The antibody recognizes PIN, as wellas prostate cancer cells. The antibody thus provides a tool for earlydetection of melanoma and prostate carcinoma. The antibody shows littleor no cross-reactivity with normal tissues, either melanocytes orprostatic tissue and thus can provide accurate information regardingtumor location and provide a means for targetting cancer cells.

[0020] The Xiphophorus fish melanoma model is well established as amodel for human malignant melanoma. In both, the melanomas are made upof the same cell type and are of the same developmental origin, theneural crest. The stages of melanoma progression which have beencharacterized for melanomas in humans are similar to those found infish. One particular type of human melanoma, familial subcutaneousmalignant melanoma is clustered in families, thereby indicating agenetic basis. Genes identified in the fish as being relevant to pigmentcell development and/or melanoma formation can therefore be used asprobes to isolate the human counterpart from genomic or cDNA libraries.Genes also can be used in raising antibodies against antigens found onhuman melanoma cells.

[0021] Several genetic factors can be identified which mediate theprogression of a normal melanin-bearing pigment cell into a malignant,metastatic melanoma cell. One complex locus encompasses geneticinformation for the formation and location of macromelanophores and alsofor melanoma permissiveness; the term “macromelanophore” refers to thelarge melanophores found in Xiphophorus which are larger and have moremelanin pigment than melanocytes in the human. To identify oncogenerelated genes on the chromosome carrying the complex locus, theXiphophorus fish genome can be screened with conserved oncogene probes.Genetic linkage analysis is then used to identify genes which areclosely linked to the complex locus and thus a candidate for any of theencoded information in the complex locus, for example, for melanomapermissiveness, and then to differentially screen for those genes foundonly in cells which produce the pigment cell giving rise to melanomas.Examples of such genes are the erb-B related gene, and the src (Roussarcoma virus) oncogene. Genomic and cDNA clones are isolated andpartially sequenced: and RNA expression studies carried out to identifygenes which are preferentially expressed in tissue containingmelanophores and which are highly expressed in the fish melanomas. Usingthis methodology genes which are specific for melanomatosis pigment cellgrowth can be identified. The gene product, or a portion thereof then isused to generate monoclonal antibodies. In order to obtain a highlyspecific antibody, the deduced amino acids encoded by the gene can beevaluated to identify a portion of the expression product most likely tobe highly antigenic such as the extracellular domain of a surfacereceptor. DNAs encoding the human mrk protein from melanoma and fromprostate cancer can be identified in a variety of ways. For example,genomic or cDNA libraries from prostatic cancer cells are screened withdetectable enzymatically-or chemically-synthesized probes, which can bemade from DNA, RNA, or non-naturally occurring nucleotides, or mixturesthereof. Probes may be enzymatically synthesized from DNAs from theXmrk-protein for normal or reduced-stringency hybridization methods.Oligonucleotide probes also can be used to screen prostatic cancer andmelanoma cells and can be based on sequences of known epidermal growthfactor receptors, including conserved sequences, or on peptide sequencesobtained from the purified protein. Hmrk protein from prostatic cancerand melanoma cells. Oligonucleotide probes based on amino acid sequencescan be degenerate to encompass the degeneracy of the genetic code, orcan be biased in favor of preferred human codons. Oligonucleotide probesencoding a consensus sequence which includes the amino acids DFPAL ispreferred. Oligonucleotides also can be used as primers for PCR fromreverse transcribed mRNA from prostatic cancer cells or melanoma cells;the PCR product can be the full length cDNA or can be used to generate aprobe to obtain the desired full length cDNA. Alternatively, the Hmrkprotein can be entirely sequenced and total synthesis of a DNA encodingthat polypeptide performed.

[0022] Once the desired genomic or cDNA has been isolated, it can besequenced by known methods. It is recognized in the art that suchmethods are subject to errors, such that multiple sequencing of the sameregion is routine and is still expected to lead to measurable rates ofmistakes in the resulting deduced sequence, particularly in regionshaving repeated domains, extensive secondary structure, or unusual basecomposition, such as regions with high GC base content. Whendiscrepancies arise, resequencing can be done and can employ specialmethods. Special methods can include altering sequencing conditions byusing different temperatures; different enzymes; proteins which alterthe ability of oligonucleotides to form higher order structures; alterednucleotides such as ITP or methylated dGTP; different gel compositions,for example adding formamide; different primers or primers located atdifferent distances from the problem region; or different templates suchas single stranded DNAs. Sequencing of mRNA can also be employed.

[0023] The sequence also can be evaluated to identify a unique aminoacid sequence, i.e., a portion of the polypeptide which is not found inother proteins using data bank comparisons and computer modeling. Apeptide containing the amino acid sequence and optionally any additionalresidues desirable for coupling to a carrier for immunization is thenprepared. The oligopeptides combine the desired amino acid sequences insubstantially pure form. Thus, usually the subject composition is atleast 80 mole percent, usually at least about 90 mole percent of theparticular oligopeptide or mixture of oligopeptides which come within aparticular formula.

[0024] The subject compounds are made in conventional ways which can beemployed for the production of oligopeptides. Techniques include usingautomatic peptide synthesizers, employing commercially availableprotected amino acids. Alternatively, recombinant DNA technology may beemployed, by synthesizing according to conventional procedures theappropriate nucleotide sequence, joining the sequence to an appropriatereplication vector, transforming a host cell and cloning and growing thetransformed host cell to produce the oligopeptides of interest which maythen be isolated.

[0025] For preparation of antibodies, the subject compounds areconjugated to an immunogenic carrier, for example antigen proteins, toact as a hapten for production of antibodies specific for an epitope onmelanoma or prostatic carcinoma cells. Various proteins are employed asantigens which are not endogenous to the host. Commonly employedantigens are the albumins, globulins, keyhole limpet hemocyanin, or thelike. Haptenic conjugates to antigens are well known in the literatureunder and are amply exemplified in a wide variety of patents. See forexample, U.S. Pat. Nos. 4,156,081, 4,069,105 and 4,043,989.

[0026] The haptenic antigen conjugates are used in accordance withconventional ways to immunize a mammal, for example, rat, mouse orhigher mammal, for example, primate, including human in accordance withconventional procedures. See, for example, U.S. Pat. Nos. 4,172,124,4,350,683, 4,361,549, and 4,464,465. Monoclonal antibodies can beproduced as a result of hybridoma formation and expression by thehybridoma whether in culture or present as ascites. Hybridomas areprepared by fusing available established myeloma lines, for example,SP2-0. NS/1, AG8.6.5 3, etc. with peripheral blood lymphocytes, forexample, splenocytes or other lymphocytes of the immunized host. Themonoclonal antibodies can be any mammalian species, including murine,rabbit, human or the like, or combinations thereof, such as chimericantibodies, having a human constant region and a mouse or othermammalian source variable region. The antibodies can be any class orsubclass, such as IgA, IgD, IgG, IgM, and may include IgG1, 2a, 2b, or 3or the human equivalents thereof. Methods for preparing monoclonalantibodies are well established. See, for example, MonoclonalAntibodies, eds. Roger H. Kennett, Thomas J. McKearn, Kathleen B.Bechtol, Plenum Press New York. 1980 ; Nature (1975) 256:495-497; U.S.Pat. Nos. 4,271,145; 4,196,265; 4,172,124, 4,195,125; 4,262,090; and4,294,927. A monoclonal antibody fragment, such as Fab, F(ab)′₂, Fv, arecombinant variable region, or the like may also find use. Theresulting immortalized B-lymphocytes, for example hybridomas,heteromyelomas, EBV transformed cells etc., are then selected, clonedand screened for binding to the subject epitope.

[0027] Screening for antibodies which have the desired specificity canbe performed using any of a variety of methods. For example, culturedcells that express a protein epitope on the cell surface are incubatedwith radiolabelled antibody at various dilutions in the presence orabsence of unlabelled antibody. Following removal of the incubationmedium and washing of the cells, the cells are treated with detergent,such as 0.1% SDS, to solubilize the bound protein and aliquots of theresulting solution are counted. The counts from the cultures incubatedwith both radiolabelled and unlabelled antibody are subtracted from thecorresponding cultures containing only labelled antibody. The data thenare analyzed, for example, using non-linear, least squares, curvefitting routine as provided by the program Systat 5.2, to obtain theapparent affinity constant and number of binding sites. Alternatively,flow cytometry and immunocytochemistry can be used to identify thebinding of antibody to cells which express the epitope on their cellsurface. Confirmation that a particular antibody is binding to theoutside of the cell is obtained by immunohistochemical studies, whereinstaining is limited to the outer surface of the cells.

[0028] The subject immortalized B-lymphocytes or other cells, forexample T-cells, which provide receptors specific for the subjectepitope, can be used as a source of DNA, either genomic or cDNA, forexpression of the ligand heavy chains of the receptors in prokaryotes oreukaryotes. The resulting products may then be used as receptors forbinding to the subject epitopes.

[0029] The epitopes are characterized as being present on melanoma cellsand essentially absent from normal melanocytes and most of the malignantcells. The antigen binds specifically to the monoclonal antibody (XMEL)12f3.2 and has an apparent molecular weight of about 170 KDal in the PSMcell line. Once antibodies are available which are specific for theepitope, the antibody can be used for screening for different antibodiesfrom the same or a different host which bind to the same epitope byemploying the subject antibody 12f3.2 or antibodies prepared to theepitope which cross-react with 12f3.2. Monoclonal antibody 12f3.2 issecreted by hybridoma cell line 12f3.2 and is described in U.S. Pat. No.5,605,831

[0030] The antibodies find use in diagnosis, with tissue employingcytology, with lysates of tissue or in detecting the subject epitope inblood or serum. A wide variety of techniques and protocols exist fordetecting an antigen in a sample suspected of containing the antigen.Conveniently, the presence of the epitope can be determinedimmunologically by applying conventional immunoassays or histochemicalstaining techniques using antibodies reactive with the epitope expressedon the cell surface and/or shed into the blood. Protocols involve a widevariety of labels, which labels include radio-nuclids, enzymes,fluorescers, fluorescer-quencher combinations, chemiluminescers,magnetic particles, radiopaque dyes, and the like. These labels can bedirectly conjugated to the monoclonal antibody through a variety ofcovalently bonded linking groups and functionalities. Some of thetechniques involve having one of the members of the antigen-antibodycomplex bound to a support, such as a particle or vessel wall, whileother of the assays are performed in solution without a separation step.In a number of assays, the antibody need not be labeled, such as in ahemagglutination assay, or where anti-immunoglobulin is employed and theanti-immunoglobulin is labeled, so as to provide for indirect labelingof the subject monoclonal antibody. Assays which can be employedincludes assays such as ELISA, RIA, EIA, FIA (Frye, et al., Oncogene4:1153-1157, 1987) and the like.

[0031] Often, a sample is pretreated in some manner prior to performinga screening assay, generally immunoassay. A wide variety ofimmunological assay methods are available for determining the formationof specific antibody antigen complexes. Numerous competitive andnon-competitive protein binding assays have been described in thescientific and patent literature and a large number of such assays arecommercially available. It is well within the skill of one skilled inthe art to perform such screening. Sample preparation will varydepending on the source of the biological sample. Cell or tumors andother tissue samples may be prepared by lysing the cells. Serum samplestypically can be prepared by clotting whole blood and isolating thesupernatant in accordance with well known methods.

[0032] For diagnosis of melanoma. PIN or prostatic carcinoma, biopsyspecimens are the most likely source of samples for analysis.Conventional immunohistochemical staining techniques also can be usedfor detecting the epitope in tissue samples. For example, the tissuesample may be fixed in formalin or other standard histologicalpreservatives, dehydrated and embedded in paraffin as is routine in anyhospital pathology laboratory. When paraffin embedded preparations areused, the antigen should be evaluated to determine whether thepreparation denatures the antigen for analysis. Sections are cut fromthe paraffin embedded material and mounted on glass slides or thesections are prepared from cryo-preserved tissue. Alternatively,cytological preparations can be used. For example, cells from the tissuesample can be fixed on a slide, typically by exposure to formalin in abuffer at physiologic pH, followed by suspension in acetone andpelleting onto gelatin-coated slides by centrifugation. The cellularantigen can be localized, either by exposure to labeled antibody or byexposure to unlabeled antibody and a labeled secondary antibody. Theamount of the cell surface protein or antigen in the sample is directlyproportional to the amount of bound label.

[0033] Biological fluids such as semen, serum, urine, saliva and sweatalso may be assayed for the presence of the epitope as a way ofmonitoring for the presence or recurrence of a melanoma or prostatictumor, particularly metastatic cancers. Whole body imaging techniquesemploying radioisotope labels can be used for locating melanomas, orprostatic carcinoma, both primary tumors and tumors which havemetastasized. The antibodies of the present invention, or fragmentsthereof having the same epitope specificity, are bound to a suitableradioisotope, typically technetium-99, ¹²³iodine, ¹²⁵iodine, or¹³¹iodine, or a combination thereof, and administered parenterally. Forprostatic cancer, administration preferably is intravenous. Highspecific activity labelling of antibodies or fragments withtechnetium-99m is described for example in U.S. Pat. No. 5,317,091, U.S.Pat. No. 4,478,815, U.S. Pat. No. 4,478,818, U.S. Pat. No. 4,472,371,U.S. Pat No. Re. 32,417, and U.S. Pat. No. 4,311,688. Thebio-distribution of the label is monitored by scintigraphy, andaccumulations of the label are related to the presence of melanoma cellsor prostate cancer cells. Whole body imaging techniques are described inU.S. Pat. Nos. 4,036,945 and 4,311,688. The disclosures of the citedpatents are incorporated herein by reference. Other examples of agentsuseful for diagnosis and therapeutic use which can be coupled toantibodies and antibody fragments include metallothionein and fragments(see, U.S. Pat. No. 4,732,864). These agents are useful in diagnosisstaging and visualization of melanoma and prostatic cancer so thatsurgical and/or radiation treatment protocols can be used moreefficiently.

[0034] Monoclonal antibodies can be used in other ways than binding tothe subject epitopic site. Monoclonal antibodies to the epitopic sitemay in turn be used as antigens for the production of monoclonalantibodies specific for the idiotypic side of the monoclonal antibody tothe cell surface receptor, for example 12f3.2. The anti-idiotypicmonoclonal antibody can be used to detect the presence of antibodies ina host to the cell surface receptor, where the monoclonal antibody tothe cell surface receptor and the physiological fluid to be diagnosedare from the same host. For example, where the monoclonal antibody tothe cell surface receptor is a human antibody or a humanized antibody,then the monoclonal antibody to the cell surface receptor can be used asan antigen to make monoclonal antibody specific for the human idiotypefor the cell surface receptor, which monoclonal antibodies can be usedto detect antibodies to the cell surface receptor which are present in ahuman physiological fluid, for example blood or serum. Theanti-idiotypic monoclonal antibody can be made in any host, for examplerodent, more particularly rat or mouse.

[0035] In addition, the conformation of the idiotype of theanti-idiotypic monoclonal antibody resembles the epitope of the cellsurface receptor and thus can serve as an antigen in competition withthe cell surface receptor epitope. To that extent, the idiotypicmonoclonal antibody can serve as a vaccine in inducing an immuneresponse to the cell surface receptor epitope different from the immuneresponse obtained with the cell surface receptor. Furthermore, theanti-idiotypic monoclonal antibody can serve as a reagent as a ligandwhich is competitive with the cell surface receptor. The monoclonalantibodies also can be used as a means of purifying the cell surfacereceptor from melanoma cells and prostatic cancer cells, for example incombination with a solid support, to form an affinity matrix.

[0036] The specificity of the monoclonal antibodies makes them useful astargeting agents for human melanoma cells or prostatic cancer cells. Forexample, the antibody can be coupled to a cytotoxic agent using methodsknown to those skilled in the art. For example, see U.S. Pat. No.4,590,071 and U.S. Pat. No. 5,055,291, which disclosures areincorporated herein by reference. T-cell therapy also may be used, forexample, see Rosenberg, New England Journal of Medicine 316: 789, 1987,which disclosure is incorporated herein by reference.

[0037] A composition of the invention for use in vivo generally willcontain a pharmaceutically acceptable carrier. By this is intendedeither solid or liquid material, which can be inorganic or organic andbe of synthetic or natural origin, with which the active component ofthe composition is mixed or formulated to facilitate administration to arecipient. Any other materials customarily employed in formulatingpharmaceuticals are suitable.

[0038] The subject compositions can be provided as kits. The kitsinclude an antibody or an antibody fragment which binds specifically toan epitope on melanoma or prostatic tumor cells, particularly anantibody produced by hybridoma cell line 12f3.2, and means for detectingbinding of the antibody to its epitope on melanoma or prostatic tumorcells, either as concentrates (including lyophilized compositions),which may be further diluted prior to use or at the concentration ofuse, where the vials may include one or more dosages. Conveniently,where the kits are intended for in vivo use, single dosages may beprovided in sterilized containers, having the desired amount andconcentration of agents. Where the containers provide the formulationfor direct use, usually there will be no need for other reagents for usewith the method, as for example, where the kit contains a radiolabelledantibody preparation for in vivo imaging and/or diagnosis.

[0039] The following examples are offered by way of illustration and notby way of limitation.

EXAMPLES Example 1 Preparation of Melanoma-Specific Antigen

[0040] The Xiphophorus fish genome was screened with conserved oncogeneprobes to identify oncogene-related genes on the chromosome carrying thecomplex locus which encompasses genetic information for the formationand location of macromelanophores and melanoma permissiveness. Toisolate the genes of the major loci, oncogene probes were used toidentify restriction fragment polymorphisms (RFLPs) as markers for theseloci. RFLPs were found for genes similar to the src and the erb-B genes(Vielkind and Dippel, Canadian Journal of Genetics and Cytology, Vol.26:607-614 (1984)). The latter gene appears to be closely linked to thesex-linked pigmentary locus and is considered to be a candidate gene forsome of the information contained in this locus.

[0041] Using the erb-B oncogene probe, a genomic phage library wasscreened and several positive clones were isolated. The clones werecharacterized by restriction enzyme mapping, which yielded five clonesthat encompassed the same restriction fragments as those identified ingenomic southern blots using the erb-B probe. Probes from theseXiphophorus clones were used to screen two cDNA libraries yieldingtwenty clones, one of which carried an insert corresponding to the RNAfragment detected in a Northern blot analysis of the RNA from the PSMcell line. Restriction mapping of this clone as well as partialsequencing revealed that the identified gene is probably identical tothe published Xmrk gene (Wittbrodt et al., 1989. Nature 341:415-421)

[0042] Differential PCR analysis with total RNA from testis, ovary,liver, kidney, brain, eye, skin, skin areas with melanophores, andmelanomas using mrk-specific primers revealed that mrk is preferentiallyexpressed in tissue containing melanophores and is highly expressed inthe melanomas. This and the fact that this gene is found only in fishthat can produce the cells giving rise to melanoma make it a specificgene for melanomatosis pigment cell growth.

[0043] Comparisons of the mrk sequence were done with sequencescontained in the latest available Genebank and EMBL Genebank computerprograms and its deduced amino acid sequence showed that the putativeprotein belongs to the family of receptor-tyrosine kinases which includean extracellular domain, a transmembrane domain and a cytoplasmictyrosine kinase domain; the protein is most closely related to the humanepidermal growth factor receptor (FIG. 1) but contains unique sequencesnot present in the epidermal growth factor receptor. The cytoplasmictyrosine kinase domains are highly conserved domains and thetransmembrane domain is unlikely to be highly antigenic. Therefore, theextracellular domain was further evaluated to find a portion of thatdomain which is not found in other proteins such as the epidermal factorreceptor protein and for which high antigenicity can be assumed.

[0044] A computer modeling (Surfaceplot-Synthetic Peptides, Inc.; seeParker et al., Biochem 25:5426-5432, 1985) of the putative mrk-receptorprotein was done to predict the antigenicity of portions of thisprotein. An exposed sequence of 10 amino acids (L-F-R-S-E-D-Q-S-I-E)(SEQ ID NO: 1) of the extracellular domain met this demand. The aminoacid sequence of this portion of the mrk-protein also avoids anysimilarity to the amino acid sequences of the true epidermal growthfactor receptor of fish or human origin and according to an extensivesearch in the gene/protein data banks, no significantly similar aminoacid sequences exist in other proteins. This sequence, therefore,appears to be rather specific for the novel receptor; a peptide wastherefore synthesized and used for the generation of antibodies (FIG.1). For coupling to the hapten, the peptide was synthesized with acysteine residue at the N-terminus. The peptide was coupled to thehapten KLH (keyhole limpet hemocyanin, Pierce Chemical Co.), via SMCC(succinlmidyl 4-(N-malemido-methyl)cyclohexane-] carboxylate, PierceChemical Co.) (see Sutcliffe et al., 1980; Sela, Synthetic Vaccines Vol.1 R. Amon (Ed.), CRC Press, Boca Raton, Fla., pp. 85-92, 1987).

Example 2 Preparation of Antibodies to Mrk-Receptor Protein Fragment

[0045] Immunization

[0046] The hapten-peptide conjugate was mixed 1:1 with Freund's completeadjuvant and 0.8 ml injected intraperitoneally (ip) into each of threeFischer rats (125-150 g body weight) at a concentration of 0.5 mg/ml.After 14 days, a second ip injection of the KLH-peptide conjugate wasgiven, and then 4 times at 14 day intervals the uncoupled peptide at aconcentration of 0.5 mg/ml was injected. At that time, a positiveimmunoresponse of the serum of the rats was identified by standard ELISAmethodology using cells from the Xiphophorus fish melanoma cell line(PSM line) which show a high expression of the mrk gene as positive andthe Xiphophorus fibroblastoid cell line (XGI line) as negative controlas was the rat preimmune serum; staining was done using goat anti-ratIgG (H+L) (Jackson Immun. Lab.) as secondary antibody conjugated tohorseradish peroxidase, and OPD (o-phenylenediamine dihydrochloride,Sigma) as the chromogenic substrate. Another ip immunization with thepeptide at a higher concentration (0.8 mg/ml) then was done followed byan intravenous injection of the peptide (0.8 mg/ml) without Freund'sadjuvant 3 weeks later. Four days later monoclonal antibody productionwas started by fusing 4×10⁸ spleenocytes from the rat which gave thestrongest immunoresponse to 0.8×10⁸ SP2/mouse myeloma cells. Fourteen96-well microtitre plates were seeded with a spleen cell density of1.25×10³ cells/well. Cells were grown for 7 days in HAT-DMEM (TFL MediaPreparation Service, Vancouver, B.C.) supplemented with 50 μl/ml ofInterleukin-8.

[0047] Isolation of Clones

[0048] Screening of monoclonal producing hybridoma cells was done onterazaki plates coated with cells fixed in 0.05% glutaraldehyde from thePSM melanoma cell line as positive control and as negative controls withcells from the XGI cell line, from a gonadal cell line of trout, withfibroblasts and bone marrow cells from human; staining was done as notedabove. Cells from 28 wells which yielded a positive signal with the PSMcells were transferred from the microtitre plates into 1 ml of HT-DMEMin 24-well plates and after 3-6 days plated in methylcellulose.Isolation of individual pure hybridoma clones was done by picking atleast 12 individual colonies derived from the original positive cells,transferring them into 96-well microliter plates and testing again forpositive reactivity with the PSM cells and also with the peptide; asnegative controls the cells mentioned above were also tested. Threeclones were isolated which produced monoclonal antibodies that reactedpositively with the peptide and the fish melanoma cell line butexhibited no reactivity with the other cells.

[0049] Two other clones, 10A3 and 3B7 reacted less strongly and threeclones, 5C1, 6E4, and 14C7, reacted not only with the peptide and thefish melanoma cell line but also with a trout cell line, making themmore general.

[0050] Typing of Antibody

[0051] Supernatants of the three positive hybridoma clones weresubsequently reacted with cultured PSM as well as XGI cells fixed on theculture dishes with 4% paraformaldehyde (20 min, RT); staining was doneusing anti-rat IgG(H+L) conjugated to horseradish peroxidase (JacksonImmun. Lab.) and DAB (3,3′-diaminobenzidine. Sigma) as chromogenicsubstrate or with goat anti-rat F(ab)′₂ FITC-coupled secondary Ab(Cappel Lab.). The clone 12f3.2 produced the strongest signal with thePSM cells and we thus concentrated on it first. Typing of this mAb usinga Bio-Rad kit which is a mouse typing kit but also has cross-reactivitywith rat antibodies revealed that the monoclonal antibody 12f3.2 is anIgM(k).

[0052] Immunostaining of Cells from Xiphophorus PSM Melanoma Cell Line

[0053] To further verify the specificity and also identify the locationof the antigen recognized by the peptide-specific mAb, cultured cells ofthe PSM cell line were fixed and stained (see above) with supernatantsof the mAb producing clone 12f3.2. The PSM line contains all stages ofpigment cell differentiations: spindle-shaped premelanocytes, youngmelanocytes with fine few dendrites, polydendritic adult melanocytes,and finally also the large melanophores with broad dendrites which arefully loaded with brown-black melanin pigment. Staining however, wasfound only in the younger stages, the melanocytes, at the contact sitesof their dendrites, on the edges concentrated in spots and in areaswhere the cells narrow towards the dendrites. This staining pattern istypical for a cell surface receptor or cell contact type antigen. Thisis in keeping with data we have deduced from the nucleotide sequence,i.e. that the putative mrk protein is a cell surface receptor. Thisstaining behavior is more prominent in vitally stained cells and usingthe FITC anti-rat IgG conjugate.

Example 3 Characterization of Antibody Specificity for Melanoma TissuemAb Reacts with Pigment Cells from Normal and Melanoma Tissue InXiphophorus

[0054] While the culturing of melanoma cells can yield valuableinformation, the classification of the disease state must be done onsectioned material. In order to test the suitability of the produced mAb12f3.2, cross-sections from frozen as well as paraffin embedded materialwere stained using the avidin-biotin procedure (ABC staining kit, VectorLab.) and AEC (3-amino-9-ethylcarbazole, Sigma) as chromogenicsubstrate. So far we have obtained with mAb 12f3.2 supernatant (1/50 inPBS) a positive signal in frozen positioned and acetone-fixedXiphophorus dorsal fin material which contains in the dermis fullydifferentiated melanophores and immature, non-pigmented precursorsunderlying the differentiated cells. Interestingly, staining was notobserved in the melanophores but in the precursors. Sections of melanomatissue stained positive in areas representing tumor growth and in areaswhich are composed of spindle-shaped, lightly pigmented cells.

[0055] mAb Stains Human Melanoma Cells but not Normal Melanocytes InVitro

[0056] The encouraging results of the detection of melanoma cells fromour model led us to further test the mAb by immunostaining humanmelanoma cell lines derived from patients from whom B-cells were alsoavailable as a control. The cells in the culture dishes were fixed with0.4% paraformaldehyde for 30 min. at RT and then immunostained asdescribed above for cultured fish cells. All three melanoma cell linesKZ-2,-13,-28 stained positively but each in a characteristic way. Thespindle-shaped, dendritic KZ-2 cells exhibited an evenly distributedstaining with some spots of more concentrated staining, the dendritic topolydendritic KZ-13 cells exhibited the staining in the cell's body andat the start of the dendrite, and the larger KZ-26 cells which havebroad dendrites and tend to conglomerate show strong staining in thecenters of these concentrations presumably where their dendrites contacteach other. The B-cells of the patients did not show any staining at allnor did the cultured melanocytes derived from human foreskin. Thehistory of the human melanoma cells are not quite clear with regard toclassification of the described stages above, i.e. radial, verticalgrowth phase, metastatic phase. However, the KZ-2 cells show thefastest, KZ-13 slower and the KZ-26 cells an even slower growth. Thus,the mAb appears to yield a characteristic staining of melanoma cells ofvarious growth potentials.

[0057] mAb Reacts with Human Melanoma Tissue

[0058] The most important question to answer was whether or not the mAbspecifically recognizes human melanoma tissue. Sections from threeprimary and four metastatic acetone-fixed and paraffin-embedded humanmelanoma specimens obtained from six different patients were stainedwith the mAb 12f3.2 supernatant diluted 1/50 with PBS as described abovefor the sections of fish tissue; all melanomas showed positive staining.In the primary melanomas, nests of stained (positive) melanoma cellswere observed while in the metastatic tumors the positive cells weremore dispersed; two of the metastatic tumors of the same patient showedstrong staining that appeared as grainy spot indicative of antigenlocated on the surface of the melanoma cells. The healthy skin,including melanocytes and other cell types within the melanoma, e.g.lymphocytes, did not show any staining.

[0059] Identification of Melanoma Related Characteristics by WesternBlotting

[0060] Another possibility to identify melanoma specificity is toanalyze electrophoretically separated proteins on nitrocellulose blots.Therefore cells of the PSM and KZ melanoma cell lines were lysed andseparated and blotted according to standard conditions and the blotsimmunostained with the mAb. Only the melanoma showed a positive signal;the PSM cells revealed a weak signal at 170 KD which would be expectedfrom the predicted amino acid sequence and a strong signal of approx. 70KD which may be a degradation product of the activation and signaltransduction of the mrk-receptor tyrosine kinase (see Ullrich andSchlessinger, Cell 61:203-212, 1990). Signals in very similar sizeranges were detected in the human melanoma cells. No signal at all wasfound in lysates derived from the control XGI cell line, which is to beexpected as these cells do not express the mrk gene.

[0061] A rat monoclonal antibody was produced against a syntheticpeptide which represents a putative highly antigenic portion of a novelreceptor tyrosine kinase protein. The protein is coded by a melanomapigment cell specific gene which was cloned from Xiphophorus fish.Xiphophorus represents an animal melanoma model which is wellestablished as a model for human melanoma formation.

[0062] The mAb recognizes the prepigment cells in situ, theundifferentiated melanocytes of a Xiphophorus melanoma cell line andalso the melanoma cells in growth areas of Xiphophorus melanomas. Italso reacts characteristically with cultured human melanoma cells ofdifferent growth potentials and with primary and metastatic secondarymelanomas in situ. The staining pattern of secondary melanoma cells hasa grainy appearance. The mAb does not react with normal human culturedmelanocytes nor with those that are located in the normal skin adjacentto the melanomas. In conclusion, the mAb can be used as a tool forhistochemical characterization of melanoma cells in patients forimmunodiagnosis of both primary and metastic melanomas, and fortreatment of melanomas by immunotherapy.

Example 4 Antibody 12f3.2 Reactivity in Human Melanomas

[0063] To test the antibody 12f3.2 (XMEL) reactivity in human melanomaand to answer the question whether the XMEL antigen is related to theearly or later stages of melanoma progression, 136 specimens coveringthe stages of progression of malignant melanoma were immunostainedaccording to the protocols in Example 3. XMEL reactivity can besummarized as follows: (1) weak reactivity was found in 2/18 (18%)common acquired nevi, (2) strong XMEL staining was observed in 8/21(38%) dysplastic nevi which is believed to be the precursor of malignantmelanoma, (3) similarly strong reactivity was seen in 53/60 (88%)primary melanomas and (4) staining was less consistent in metastaticmelanomas ranging from very strong to weak and absent in some of them,23/37 (62%) were XMEL positive. These data suggest that the antibodyrecognizes an antigen that is related to the early events (primarymelanomas) rather than the later stages of progression to the metastaticphenotype (metastatic melanomas). In another cohort study of high riskpatients with deep melanomas, XMEL immunoreactivity was found notrelated to patient outcome while Breslow's depth of invasiveness was(Breslow's depth is considered to be the most reliable, singleprognosticator but is not always applicable). The findings also indicatethat XMEL antibody recognizes early stage rather than late stage ofmelanoma.

[0064] The reactivity of XMEL with the different progression stages iscompletely different from that of antibodies against the EGFR (epidermalgrowth factor receptor) protein. The resultings of antibodies againstEGFR protein show an increase in reactivity with an increase inprogression and the highest reactivity is found in metastatic melanomas;whereas the results of XMEL antibody show the highest reactivity withprimary melanomas. Therefore, the XMEL antibody does not recognize onlythe EGFR protein.

Example 5 Specificity of XMEL Antibody

[0065] This study was performed to determine the specificity of the XMELantibody. A screen of nearly 100 non-melanocytic malignancy samples weretested by immunostaining as described in Example 3. There was virtuallyno cross reactivity with any other cancer type; XMEL immunostaining wasobserved on all 4 carcinomas of the prostate. Fourteen more samples werestudied. All specimens exhibited strong XMEL reactivity on the membranesof cells representing high grade PIN, especially on cells at the edgesof invasive carcinoma in PIN and in cancerous glands in the vicinity ofPIN. These are sites presumed to be characteristic of cells which havechanged into invasive malignant cells.

Example 6 Specificity of Antibody 12f3.2 for Prostate Cancer

[0066] The initial study was performed on biopsies from radicalprostatectomies (15 cases), all these cases fulfilled the criterion thatno treatment prior to the operation was given to the patient which couldotherwise cause XMEL antibody reactivity. In the studies, it wasobserved that the XMEL antibody reacted with high grade prostaticintraepithelial neoplasia (hg PIN) and invasive prostatic cancer cells;hg PIN is considered to represent the precursor of the malignantprostate cancer cell. We then extended these studies to further 34 casesof radical prostatectomies and normal tissue samples (Table 1 and 2).TABLE 1 Immunohistochemical reactivity of the monoclonal antibody XMELfrom the hybridoma clone 12f3.2 in biopsies from radical prostatectomiesand samples from normal tissues A. Prostatectomies XMEL % of XMELstaining XMEL Biopsy from positive intensity on staining in patientcancer cells cancer cells normal gland Control A-1 100 3+-4+ − − A-2 2l+-2+ − − A-3 60 3+-4+ − − A-4 60 3+-4+ − − A-5 50 3+-4+ − − A-6 95 4+ −− A-7 70 3+ − − A-8 2 1+ − − A-9 2 2+ − − A-10 2 2+-3+ − − A-11 40 2+-3+− − A-12 5 1+ − − In these biopsies, high grade prostaticintraepithelial neoplasia (hg PIN) was not observed. B. Normal tissueSample # Tissue Type Control A-13 normal prostate − A-14 seminal vesicle− A-15 liver − A-16 muscle − A-l7 kidney − A-18 lung − A-19 colon − A-20skin −

[0067] TABLE 2 Immunohistochemical reactivity of the monoclonal antibodyXMEL from the hybridoma clone 12f3.2 in biopsies from the radicalprostatectomies from a cohort of cancer patients. XMEL XMEL SMEL % ofstaining staining XMEL intensity intensity on staining Biopsy positiveon cells in hg in from cancer cancer PIN, if normal patient cells cellspresent gland Control B-1 0 − − B-2 0 − − B-3 10  1+ − − B-4 0 1+ − −B-5 0 1+ − − B-6 0 − − B-7 no cancer 1+ − − B-8 0 − − B-9 0 − − B-10 10 1+ − − B-11 0 − − B-12 0 1+ − − B-13 0 1+ − − B-14 0 − − C-1 0 1+ − −D-1 30  1+ − − D-2 10  1+ 1+ 1+ − D-3 20  1+ 1+ − − D-4 30  1+ 1+ − −D-5 40  1+ 3+ − − D-6 5 1+ 1+ − − D-7 0 − −

[0068] In Table 1B, we presented results from normal tissue samples,because for a possible immunotherapy with this antibody it is necessaryto establish that the antibody shows no cross-reactivity with normaltissues of the patient. As can be seen from Table 1, all prostatecancers stained positive with the antibody although with variousintensities and variable numbers of the cancer cells, while the normalgland component was negative as were the normal tissues including thesections from non-cancerous, normal prostate.

[0069] The data shown in Table 2 pertain also to the question ofspecificity but represent the initial data on the question of prognosis.These samples were chosen from a cohort of patients from whom tissueblocks had been obtained and stored for several years. The reactivitywith these biopsies is less strong, which most likely is due to the factthat these are sections from old paraffin blocks which usually yield aless strong immunosignal. The fact that 14/22 cancer and/or PIN samplesstained positive shows that the antibody is specific for the malignantstages of carcinoma of the prostate.

Example 7 Immunoreactivity in Prostatic Carcinomas

[0070] A total of 115 biopsies from prostatectomies (Studies 1-4) and 10biopsies from normal glands (Study 5) were tested in the experimentusing immunostaining as described in Example 3. The specimens used instudies 1-3 are cancer of the prostate. The specimens of study 4 arenegative for cancer of the prostate. Each biopsy specimen was taken froma different individual. 91/94 (97%) of carcinoma of the prostate and52/54 (97%) of high grade PIN reacted positively with the XMEL antibody.The data are summarized in Table 3. TABLE 3 Immunohistochemicalreactivity of the monoclonal antibody XMEL in biopsies from radicalprostatectomies and normal glands XMEL # of XMEL staining XMEL staining# of intensity # of positive intensity XMEL on biopsies staining oncells in # of positive cancer having in hg hg PIN, if Study biopsiesstaining cells hg PIN PIN present 1 14 14 3+-4+ 14  14 3+-4+ 2 12 123+-4+ 0 3 68 65 2/3+-4+ 37  35 3+ 4 21  0 3  3 2+-3+ 5 10  0 0

[0071] The high incidence of XMEL antibody staining of PIN and prostatecancer suggests that the antigen and its encoding gene are instrumentalin the development of prostate cancer.

Example 8 XMEL Immunostaining of Prostate Carcinoma

[0072] To test the reactivity of the XMEL antibody on prostatecarcinoma, immunostaining of sections from paraffin-embeddedprostatectomies with XMEL antibody and biotin-streptavidin-peroxidasedetection system was performed using AEC as a chromogenic substrate asdescribed in Example 3. XMEL antibody detected surface antigen on highgrade PIN and invasive malignant cells. The results are shown in FIG. 4.FIG. 4 also shows that the best XMEL staining was found on moderatelydifferentiated carcinoma cells (Gleason grade 3) and that lessimmunostaining was found on only differentiated cells. Thus XMELreactivity may aid in distinguishing high and low risk cancers. Noreactivity was observed in the stromal component nor on the normalacini. Some membrane luminal staining was observed, indicating thatXMEL-reactive cell surface antigen is shed into the blood of prostatecancer patients.

Example 9 XMEL Reactivity in Relation to Cancer Diagnostic/PrognosticParameters

[0073] The almost 100% reactivity of XMEL with high grade PIN andprostatic cancer cells (see Example 7) strongly suggests a role for theXMEL antigen in prostatic cancer particularly in the early stages. Todetermine if XMEL reactivity is an indicator for the disease stage, 277samples for which a pathological report containing information on tumorstage, PSA level, Gleason grade, follow up, and other demographicinformation is available are obtained. Included in this cohort are 100cases with needle biopsy results which allows the comparison of XMELreactivity with the diagnosis of the physician/pathologist and thecancer that ultimately occurs. To determine whether the XMEL antigen isexpressed in clinically insignificant small volume cancers, 40 specimenswith occult prostatic carcinoma from radical cystoprostatectomy ofbladder cancer are evaluated. As controls, benign prostatic hyperplasiaand PIN of lower grades from the tissue bank are included. Strongstaining in the high grade PIN and the invasive cancer cells areexpected and the relationship between XMEL reactivity and the usualpathological/clinical parameters is determined.

[0074] Sections are prepared from these samples and immunostained asdescribed previously. XMEL reactivity is determined by two individualsin a blind study by assessing both intensity of staining and percentageof positively stained cells. Intensity of staining is scored as follows:− absent=1,+ weak=2,++ moderate=3,+++ strong=4; percentage of positivecells will be graded according to the following: 1-5%=1, 6-25%=2,26-50%=3, 51-75%=4, and 76-100% =5. Strength of reactivity is defined asintensity times percent of positive cells and intensity score over thenormal and malignant glands. The results is assessed using analysis ofvariance statistics (ANOVA). Following update of patient status, theXMEL data along with the pathologic/clinical data is analyzed using aCox proportional hazards model to evaluate whether the level of XMELactivity is a significant diagnostic/prognostic factor for theevaluation of the cancer.

Example 10 Determination of the Presence of a 170 KDa XMEL ReactiveProduct in Sera from Patients with Confirmed Prostatic Carcinoma

[0075] Sera from 120 patients with localized prostate cancer arecollected. These samples are drawn before surgery and 3 monthspost-radical prostatectomy for determination of prostate specificantigen (PSA) levels and will be used by us for western blot analysis.Pre- and post-operative levels are evaluated with pathologic stage,grade and PSA levels. To evaluate cancer specificity, serum samples fromyoung men with normal DRE and PSA levels (i.e. low risk for prostatecancer) are measured for presence of 170 KDa XMEL-positive product aswell.

[0076] In the study, the XMEL antibody is used in form of serum-freehybridoma supernatant. Alternatively, the antibody can further bepurified by affinity chromatography on an anti-k-column or a ConA-Sepharose column and fragmented by the Pierce Immuno-PureFragmentation kit. A purified antibody/fragment allows the performanceof a capture ELISA for assessing the serum protein and increases signalstrength in the assay. Statistical analysis is done as indicated above.

Example 11 Isolation of Human mrk Gene

[0077] The similarities of fish and human melanoma formation and theXMEL reactivity with both fish and human melanoma suggests that the XMELantigen expressed in human melanoma is encoded by a gene homologous tothe novel receptor tyrosine kinase detected in the fish model. However,XMEL also recognizes prostate cancer cells. While there is no obvioussimilarity between both cancers, a receptor kinase can be involved inthe formation of more than one cancer. Thus, it is possible that thesame antigen is expressed on both cancers or a different antigen sharingthe same or different epitope with a different affinity to XMEL.Therefore, the XMEL antigen could be encoded by different genes not onlybetween fish and humans but also between melanoma and prostate cancer.What is particularly striking is that XMEL appears to preferentiallyrecognize a membrane-bound antigen on cells of the precursor andintermediate and less on cells of the advanced stages in both tumors,suggesting that the detected XMEL antigen is indicative of an early oreven initiating event in tumor formation.

[0078] A common method to clone a gene from one species when thesequence from another species is known is to use degenerate primers in aPCR protocol. A dilemma arises in selecting primers—if selectedconserved in all EGFR family genes (or to the kinase domain in thekinase superfamily) the primers may lose their specificity but if theselected sequences are not conserved in the EGFR family they are lesslikely also to be conserved in the human mrk gene (Hmrk). We reasonedthe peptide sequence to induce the antibody would be reasonablyconserved in the Xiphophorus and human mrk genes and designed degenerateprimer(s) to this sequence.

[0079] In order to reduce the probability of spurious amplification butat the same time increase the probability of primer annealing and chainelongation of the proper template, the TD PCR (see Don R. H., Cox P. T.,Wainright B. J., Baker K, and Mattick J. S. Nuc. Aicd Res. 19(14):4008,1991 and Roux K. H. Biotechniques 16:812, 1994) using Xmrk primers thatencode, at the 3′end at least, amino acids which are conserved in allhuman EGFR family genes are used. By ending the primers with a codon toa conserved amino acid, minus the wobble base, it can be ensured thatthere will be a perfect match at the 3′ terminus. Furthermore, since theuse of PCR is predicated on the tolerance of mismatches, other than atthe 3′ terminus, degenerate primers are not required. Preferentialamplification of Hmrk over the EGFR family genes occurs since the Xmrkprimers have the fewest mismatches to Hmrk.

[0080] Although expression of cDNAs from various organisms as bacterialfusion proteins has been widely used to identify genes encoding proteinsto which antibodies are available, our efforts to immunoscreen bacterialcDNA expression libraries did not result in isolation of sequences thatshare homology with Xmrk sequences. Xmrk encodes a surface receptorwhich cannot adopt its native conformation in the bacterial cell and canlead to precipitation of the fusion protein making detection by the XMELin the immunoscreen impossible. It therefore is preferable to use amammalian expression system in which a receptor is presented in itsnative configuration on the cell surface. COS cells provide the mostproven and efficient mammalian expression cloning system for cellsurface proteins (Seed, 1987). This type of expression cloning has beenoptimized (Kay R. and Humphries R. K, Methods in Molecular and CellularBiology 2:254-265, 1991) and applied (Kay R., Takei F, Humphries R. K.J. Immunolol. 15:1952-1959, 1990). It has the advantage that high levelsof expression are obtained alternatively, cell surface proteins can becloned by retroviral transmission and expression of cDNA libraries. Thisapproach results in stable expression of cDNAs allowing the repeatedselection and expansion of rare cell clones expressing the targetedantigen. (Whitehead et al., Mol. Cell Biol 15:704-710, 1995).

[0081] Expression cloning in the COS cell system is performed asfollows. A cDNA library from the LNCaP cell line is established in theexpression vector pCTV85, which is a dual purpose expression vector thatis equally effective for COS cell expression or as a retroviral vector.Pooled plasmid DNA for the library is electroporated into COS cells.After a 48-72 growth period, COS cells expressing the XMEL antigen ontheir surface are isolated with the XMEL antibody by panning. Replicatedplasmids within the selected cells are cloned and re-purified. Severalrounds of electroporation and selection of expressing COS cells aretypically required to enrich for and finally isolate positive cDNAclones, identified by their ability to confer high levels of XMELreactivity on COS cells.

[0082] A library is screened via retroviruses. The plasmid DNA of thelibrary is transfected into the BOSC 23 ecotropic packaging cell line,and secreted virus is used to infect NIH 3T3 cells. After a 4 day periodof expansion, the infected cells are viably sorted with the XMELantibody, re-expanded in culture, resorted and then tested for XMELreactivity by FACS. WHen a pure XMEL reactive population is obtained,the proviral library cDNAs carried by the cells are recovered by CPR,cloned into pCTC85, and tested for their abilities to confer XMELreactivity as individual clones.

[0083] Using antibodies bound to various solid matrixes has been ageneral method to isolate antigens to high enough purity to be used inmicrosequencing of peptides. From these sequences degenerateoligonucleotides can be synthesized which allow first to clone parts ofthe gene from total RNA or cDNA libraries in a PCR approach and then thetotal cDNA of the gene. Sequencing of the cloned PCR products determinesthe encoded amino acid sequence and can be used to verify that thecorrect gene has been isolated.

[0084] The XMEL MoAb produced under serum-free conditions in hybridomaserum-free growth medium (Gibco) is covalently linked to CNBr-activatedSepharose beads (Pharmacia). Lysates from Nonidet P-40 solubilized LNCaPcells are incubated with these beads. Bound proteins are eluted byboiling in 1% SDS, concentrated by ultrafiltration, further purified bysize exclusion HPLC, if necessary, and then subjected to preparativeSDS-PAGE (Damen et al., 1996). Following Ponceau S staining oftransferred proteins on an Immobilon membrane, the protein band isexcised and the amino acid sequence determined using methods known tothose of skill in the art. The sequence is then compared with knownsequences in databanks such as Genbank. If the amino acids are not inthe data banks, degenerate oligonucleotides are generated and the cDNAfor this protein is cloned and sequenced.

[0085] To determine whether or not the XMEL antibody might bind to apeptide sequence other than that used for the generation of theantibody, short peptides displayed by phage display libraries aresurveyed for tight binding to various targets including antibodies, cellsurface receptors or enzymes by incubating the phage library with theimmobilized target, e.g. an antibody, washing away unbound phages andthen eluting the target-bound phages. Alternatively, the phage librarycan be bound to the target insolution and this complex cpatured via animmobilized secondary antibody. This allows selection for tight and lowbinding epitopes to the target by simply varying the molar ration betwenphages and target. After 3-4 rounds an enriched pool is obtained and theamino acid epitope sequences of the displayed peptides in individualclones are determined by sequencing the corresponding coding sequence inthe phage DNA.

[0086] We have used both methods in screening for tight binding epitopesto the XMEL anitbody using a phage 15 mer display library. Both methodsled to the same small population of clones with a consensus sequence ofDFPGL which is different from the XMEL peptide sequence LFRSEDQSIE (SEQID NO:3). melag 1 NARVCDFPGISCVYR (SEQ ID NO:4) 16 clones melag 2LSGSVPSLVAPYAPW (SEQ ID NO:5) 10 clones melag 3 RDLFSPCPFPGFCRQ (SEQ IDNO:6)  2 clones melag 4 MTGNSCGDFPAYCRL (SEQ ID NO:7)  6 clones melag 5WPEVDEPGLIRLPAA (SEQ ID NO:8)  3 clones melag 6 RGGSCLALSSLXLF (SEQ IDNO:9)  1 clone melag 7 GGFPGLMFGHICSD (SEQ ID NO:10)  1 clone

[0087] One strongly XMEL positive gtl1 phage expression clone, gtl1-52,from a melonama cell line contained a small insert encoding only 44amino acids that includes the consensus sequence.

[0088] The probes are used to isolate the entire transcript by screeninga LNCaP cDNA library for overlapping clones and analyzed fordifferential expression on northern blots of RNA from the LNCaP cells incomparison to cells from the DU 145 prostate cancer cell line which isnegative for XMEL reactivity. In situ hybridization is done (HumanGenome FISH Mapping Resource Centre, HSC, Toronto) to determine thechromosomal location of the gene and if it is associated with a locusinvolved in frequent chromosomal aberrations in prostate cancerdevelopment. These and sequence data will confirm that we have a genethat contributes to the genesis of prostatic carcinoma. Possibleoncogenic activation of the gene is then investigated. For example, ifthe human gene is similar to the Xmrk gene, i.e. a gene belonging to thegrowth factor receptor oncogene class, mutational, oncogenic activationcould be of various sorts ranging from overexpression to partialdeletion, insertion, rearrangement, point mutation. Gross changes areidentified by RFLP analyses of DNAs from normal blood and matchedcarcinoma material and subtler changes by comparative sequence analysisof cDNA of normal and tumor origin.

[0089] The gene is then used to find other genes functionally associatedwith it (e.g. transactivators, the ligand, downstream effectors) inorder to ascertain the biochemical pathways leading to prostaticcarcinoma.

[0090] All publications including patent applications mentioned in thisspecification are indicative of the level of those skilled in the art towhich this invention pertains. All publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

[0091] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it will be apparent to one of ordinary skill in the artthat many changes in modifications can be made thereto without departingfrom the spirit or scope of the appended claims.

1 10 1 10 PRT Unknown Description of Unknown OrganismExtracellulardomain of mrk-receptor protein 1 Leu Phe Arg Ser Glu Asp Gln Ser Ile Glu1 5 10 2 124 DNA Unknown Description of Unknown OrganismNucleotidesequence of mrk-receptor tyrosine kinase 2 cacgctgcag ctgcgctacgccaacaccat caactggagg cgcttgttcc ggtctgagga 60 ccagagcata gagtatgacgccaggactga gaatcaaacc tgcaacaacg agtgctcaga 120 ggat 124 3 41 PRTUnknown Description of Unknown OrganismAmino acid sequence ofmrk-receptor tyrosine kinase from 446-486 3 Thr Leu Gln Leu Arg Tyr AlaAsn Thr Ile Asn Trp Arg Arg Leu Phe 1 5 10 15 Arg Ser Glu Asp Gln SerIle Glu Tyr Asp Ala Arg Thr Glu Asn Gln 20 25 30 Thr Cys Asn Asn Glu CysSer Glu Asp 35 40 4 15 PRT Artificial Sequence Description of ArtificialSequenceXMEL binding epitope 4 Asn Ala Arg Val Cys Asp Phe Pro Gly IleSer Cys Val Tyr Arg 1 5 10 15 5 15 PRT Artificial Sequence Descriptionof Artificial SequenceXMEL binding epitope 5 Leu Ser Gly Ser Val Pro SerLeu Val Ala Pro Tyr Ala Pro Trp 1 5 10 15 6 15 PRT Artificial SequenceDescription of Artificial SequenceXMEL binding epitope 6 Arg Asp Leu PheSer Pro Cys Pro Phe Pro Gly Phe Cys Arg Gln 1 5 10 15 7 15 PRTArtificial Sequence Description of Artificial SequenceXMEL bindingepitope 7 Met Thr Gly Asn Ser Cys Gly Asp Phe Pro Ala Tyr Cys Arg Leu 15 10 15 8 15 PRT Artificial Sequence Description of ArtificialSequenceXMEL binding epitope 8 Trp Pro Phe Val Asp Glu Pro Gly Leu IleArg Leu Pro Ala Ala 1 5 10 15 9 14 PRT Artificial Sequence Descriptionof Artificial SequenceXMEL binding epitope 9 Arg Gly Gly Ser Cys Leu AlaLeu Ser Ser Leu Ala Leu Phe 1 5 10 10 14 PRT Artificial SequenceDescription of Artificial SequenceXMEL binding epitope 10 Gly Gly PhePro Gly Leu Met Phe Gly His Ile Cys Ser Asp 1 5 10

What is claimed is:
 1. A method for detecting the presence of melanoma or prostatic cancer in a human host, said method comprising: combining a sample from said human host with antibodies or antibody fragments which bind specifically to a peptide having the amino acid sequence depicted in SEQ ID NO:1, and detecting formation of immune complexes as indicative of the presence of melanoma cells or prostatic cancer cells.
 2. The method according to claim 1, wherein said sample is a tissue sample.
 3. The method according to claim 2, wherein said tissue sample is paraffin-embedded or cryo-preserved.
 4. The method according to claim 1, wherein said antibodies are monoclonal antibodies.
 5. The method according to claim 4, wherein said monoclonal antibodies or second antibodies to said monoclonal antibodies are conjugated to a label which provides a detectable signal.
 6. The method according to claim 5, wherein said label is a radionuclide, an enzyme, a fluorescer, a radioopaque dye, or a chemiluminescer.
 7. The method according to claim 6, wherein said radionuclide is technetium
 99. 8. The method according to claim 1, wherein said sample is a body fluid.
 9. The method according to claim 8, wherein said body fluid is blood.
 10. The method according to claim 1, wherein said antibodies bind to a surface membrane antigen of melanoma cells or prostatic cancer cells.
 11. A method for detecting high grade prostate intraepithelial neoplasia in a human host, said method comprising: combining a sample from said human host with antibodies or antibody fragments which bind specifically to a peptide having the amino acid sequence depicted in SEQ ID NO: 1, and detecting formation of immune complexes as indicative of the presence of high grade prostate intraepithelial neoplasia.
 12. A method for early detection of prostate carcinoma, said method comprising: combining a sample from said human host with antibodies or antibody fragments which bind specifically to a peptide having the amino acid sequence depicted in SEQ ID NO: 1, and detecting formation of immune complexes as an early indication of the presence of prostate carcinoma
 13. A method for treating human melanoma or prostate cancer, said method comprising: administering a therapeutic dose of a conjugate comprising a monoclonal antibody or monoclonal antibody fragment, which binds specifically to a peptide having the amino acid sequence depicted in SEQ ID NO: 1, and a cytotoxic agent.
 14. The method according to claim 13, wherein said monoclonal antibody is a humanized monoclonal antibody.
 15. A kit for screening a sample to detect melanoma or prostate cancer cells, said kit comprising: (a) an antibody or an antibody fragment, which binds specifically to a peptide having the amino acid sequence depicted in SEQ ID NO: 1; and (b) means for detecting the reaction of said antibody or said antibody fragment with said melanoma cells or said prostate cancer cells.
 16. A peptide comprising an amino acid sequence characterized as (1) present in the extracellular domain of a polypeptide encoded an the mrk gene of Xiphophorus, and (2) reactive with antibodies obtained from hybridoma cell line 12f3.2.
 17. A peptide according to claim 16, wherein said amino acid sequence is L-F-R-S-E-D-Q-S-I-E.
 18. A peptide according to claim 17, further comprising a C at either terminus of said peptide.
 19. An antigen which binds to antibodies obtained from hybridoma cell line 12f3.2.
 20. The antigen according to claim 19, wherein said antigen is derived from a human melanoma or a human prostate tumor. 